¹³C Electron Nuclear Double Resonance Spectroscopy Shows Acetyl-CoA Synthase Binds Two Substrate CO in Multiple Binding Modes and Reveals the Importance of a CO-Binding “Alcove”

Abstract

EPR and Electron Nuclear Double Resonance spectroscopies here characterize CO binding to the active-site A cluster of wild-type (WT) Acetyl-CoA Synthase (ACS) and two variants, F229W and F229A. The A-cluster binds CO to a proximal Ni (Nip) that bridges a [4Fe-4S] cluster and a distal Nid. An alcove seen in the ACS crystal structure near the A-cluster, defined by hydrophobic residues including F229, forms a cage surrounding a Xe mimic of CO. Previously, we only knew WT ACS bound a single CO to form the Ared-CO intermediate, containing Nip(I)-CO with CO located on the axis of the d z 2 odd-electron orbital (g⊥ > g|| ∼ 2). Here, the two-dimensional field-frequency pattern of 2K-35 GHz 13C-ENDOR spectra collected across the Ared-CO EPR envelope reveals a second CO bound in the d z 2 orbital’s equatorial plane. This WT A-cluster conformer dominates the nearly conservative F229W variant, but 13C-ENDOR reveals a minority “A” conformation with (g|| > g⊥ ∼ 2) characteristic of a “cloverleaf” (e.g., d x 2- y 2) odd-electron orbital, with Nip binding two, apparently “in-plane” CO. Disruption of the alcove through introduction of the smaller alanine residue in the F229A variant diminishes conversion to Ni­(I) ∼ 10-fold and introduces extensive cluster flexibility. 13C-ENDOR shows the F229A cluster is mostly (60%) in the “A” conformation but with ∼20% each of the WT conformer and an “O” state in which d z 2 Nip(I) (g⊥ > g|| ∼ 2) surprisingly lacks CO. This paper thus demonstrates the importance of an intact alcove in forming and stabilizing the Ni­(I)-CO intermediate in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation.

Theoretical Studies of Acetyl-CoA Synthase Catalytic Mechanism

“…This is true for the A-cluster without ligands as well as with CO and H 2 O ligands. These findings conform with other calculations and experimental data [37,48,51,106]. The situation is different for the methyl ligand where there is a relatively low spin density on Ni p (slightly higher for the M2 model), while the high spin density is on the iron-sulfur cubane.…”

Two ligand-binding sites in CO-reducing V nitrogenase reveal a general mechanistic principle

“…S6 ( 8 )] ( 43 ). Note that a subpopulation of this enzyme also binds a second CO to its nickel site ( 44 ). With two further reduction steps to a terminal hydride at Fe6 [fig.…”

Selective 13C labelling reveals the electronic structure of flavocoenzyme radicals

“…The radical signature is clearly asymmetric because the electron-Zeeman anisotropy, determined by the g -matrix of FADH • , exceeds the inhomogeneous spectral linewidth. By recording ENDOR spectra at different magnetic field positions in the resonance range one can now detect molecules with particular orientations with respect to the direction of the external magnetic field vector B 0 29 , 38 , 39 . For example, recording ENDOR at a magnetic-field strength B 0 corresponding to the principal value g Z of FADH • reveals hyperfine data of those molecules whose principal Z axis is aligned parallel to B 0 thus resembling data from a partially oriented sample.…”